I. Infectious Bovine Rhinotracheitis Disease
The alpha herpesvirus, bovine herpesvirus type 1 (hereinafter "BHV-1"), more commonly known as infectious bovine rhinotracheitis virus (hereinafter "IBRV") and infectious pustular vulvovaginitis virus (hereinafter "IPV"), is a direct cause of bovine disease and is a major factor in the initiation of bacterial pneumonia (Ludwig, H., In: The Herpesviruses., Ed. B. Roizman, 2:135-314 (Plenum Press: New York) (1983); Pastoret, P. P., et al, Ann. Rech. Vet., 13:221-235 (1982); Yates, W. D. G., Can. J. Comp. Med., 46:225-263 (1982); Gibbs, E. P. J. et al, Vet. Bull. (London) Part I, 47:317-343 (1977); and Gibbs, E. P. J. et al Vet. Bull. (London) Part II, 47:411-425 (1977)). Infectious bovine rhinotracheitis (hereinafter "IBR") has a worldwide distribution and is probably the single most costly disease of cattle in the United States. Respiratory IBR disease in the United States costs about $25 million. Additional losses associated with IBRV infections result from abortion storms, fatalities in newborn calves, losses in milk yield, conjunctivitis, metritis, enteritis and meningitis.
The spread of IBR in naturally and artificially bred cattle poses a serious problem, especially with the continued widespread use of frozen semen. Recurrent shedding of virus from infected bulls also constitutes a significant threat to the artificial insemination industry in the United States and to the worldwide distribution of bovine germ plasm. The incrimination of IBRV as the etiologic agent of oophoritis and salpingitis with resultant infertility and sterility adds to the seriousness of IBRV infections.
The severity of illness resulting from IBRV infections depends on the virus strain and on the age of the animal affected. After recovery from infection, animals may show signs of recurrent disease without being reexposed to the virus. Recurrent disease without reexposure occurs because the virus remains dormant, i.e., latent, in neurons of the sensory ganglia of its host and can be reactivated, even after long periods (Rock, D.L. et al, J. Gen. Virol., 67:2515-2520 (1986); Homan, E. J. et al, Am. J. Vet. Res., 41:1212-1213 (1980); and Rodriguez, L. L. et al, Am. J. Vet. Res., 45:1069-1072 (1984)). Dexamethasone treatment can also provoke recrudescence of virus shedding with or without clinical symptoms of active IBR. This suggests that reactivation and release from neuronal sites and, possibly, persistent infection of other tissues, can occur (Narita, M. et al, Am. J. Vet. Res., 42:1192-1193 (1981); Sheffy, B. E. et al, J. Am. Vet. Med. Assoc., 163:850-851 (1973); Homan, E. J. et al, Am. J. Vet. Res., 43:309-313 (1983); Ackermann, M. et al, Am. J. Vet. Res., 43:36-40 (1982); Ackermann, M. et al, Veter. Microbiol, 9:53-63 (1984); and Edwards, S. et al, Veter. Microbiol, 8:563-569 (1983)). However, reactivation of IBRV from latency can also occur spontaneously, so that cattle latently infected with field strains of IBRV represent a sporadic source of virus transmission and herd infection.
II. Known IBR Vaccines
Control of IBR is based largely on vaccination. However, vaccination may not prevent second infections by pathogenic field strains, although vaccination does increase the effective dose required to initiate second infections and it reduces the amount and duration of virus shedding. Further, vaccinated animals have less severe clinical signs than unvaccinated animals. As a result of all these factors, spread of pathogenic field strains in the herd may be reduced by vaccination.
Currently, three types of IBR vaccines are being employed: (1) killed virus vaccines; (2) subunit vaccines; and (3) modified-live virus (hereinafter "MLV") vaccines (U.S. Pat. Nos. 3,634,587 3,925,544 and 4,291,019). Killed IBR vaccines have been produced by treating IBRV with chemicals, such as formalin or ethanol, and/or physical agents, such as heat or ultraviolet irradiation. Subunit IBR vaccines have been prepared by solubilizing IBRV-infected cell cultures with nonionic detergents and purifying some of the solubilized virus proteins (Babiuk, L. A. et al, Virol., 159:57-66 (1987)). Early MLV vaccines were designed for parenteral administration and consisted of IBRV attenuated by rapid passage in bovine cell cultures or by adaptation of IBRV to porcine or canine cell cultures, by adaptation to growth in cell culture at a low temperature (30.degree. C.), or by selection of heat-stable virus particles (56.degree. C. for 40 min). Specialized types of MLV vaccines are those administered intranasally. These MLV vaccines have been attenuated by serial passage in rabbit cell cultures or by treatment of IBRV with nitrous acid followed by selection for temperature-sensitive mutants (Todd, J. D. et al, J. Am. Vet. Med. Assoc., 159:1370-1374 (1971); Todd, J. D. et al, Infect. Immun., 5:699-706 (1973); Zygraith, N. et al, Res. Vet. Sci., 16:328-335 (1974); Kucera, C. J. et al, Am. J. Vet. Res., 39:607-610 (1978); and Smith, M. W. et al, Can. Vet. J., 19:63-71 (1978)). Temperature-sensitive virus vaccines are restricted in their replication to the nasal mucosa because the temperature of the nasal mucosa is several degrees lower than that of the body. Temperature-sensitive viruses are unable to replicate at the higher temperatures of the lower respiratory tract. Hence, their growth and spread in the body is self-limiting.
The currently available IBR vaccines discussed above have serious disadvantages and have, therefore, proved unsatisfactory in commercial use. More specifically, although killed IBRV vaccines are considered to be safer than MLV vaccines, i.e., they cannot establish latency and they eliminate the problem of postvaccination shedding, they are expensive to produce, must be administered several times, and disadvantageously require adjuvants. In addition, with their use, there is the possibility of fatal hypersensitivity reactions and nonfatal urticaria. Further, some infectious virus particles may survive the killing process and thus cause disease. Moreover, animals vaccinated with killed IBRV vaccines can be infected at a later time with virulent virus. The virulent virus can establish a latent infection and can be reactivated and shed, thereby spreading infection in the herd (Frerichs, G. N. et al, Vet. Rec., 111:116-122 (1982); Wiseman, A. et al, Vet. Rec., 104:535-536 (1979); and Zuffa, A. et al, Zentralbl. Veterinaermed. Reihe B., 27:725-733 (1980)). Accordingly, although killed IBR vaccines can provide some protection against IBR, they are generally inferior to MLV vaccines in providing long term protection.
Subunit vaccines are often less toxic than killed virus vaccines, and may induce novel immunologic effects which can be of significant value. The technique for subunit vaccine preparation involves removal of capsid proteins, while leaving intact antigenic proteins that elicit protective immunity. This creates a potential for the development of serologic procedures to differentiate vaccinated from naturally infected animals. Further, subunit vaccines, though antigenic, do not contain live virus and, thus, cannot be transmitted to other animals, cause abortion, or establish latency (Lupton, H. W. et al, Am. J. Vet. Res., 41:383-390 (1980); and le Q. Darcel, C. et al, Can. J. Comp. Med., 45:87-91 (1981)). However, subunit vaccines, like killed vaccines, do not generally prevent infection and latency when animals are subsequently exposed to virulent IBRV field strains. Other disadvantages of subunit vaccines are the high cost of purification and the requirement of several injections with adjuvant.
MLV IBR vaccines have the important advantage that they produce rapid protection and activate cell-mediated and humoral components of the immune system. In the case of intranasal (hereinafter "IN") administration, localized immune responses that suppress later replication of virulent IBRV in the respiratory tract contribute significantly to protection. The local immune responses include production of interferon and IgA antibodies in nasal secretions (Kucera, C. J. et al, Am. J. Vet. Res., 39:607-610 (1978)). Extensive utilization of MLV IBR vaccines has reduced the frequency of occurrence of IBR. However, most of the available MLV IBR vaccines are not entirely satisfactory. More specifically, there is concern as to their safety, especially if the vaccine virus itself produces latency and may be shed and transmitted to susceptible cattle.
Maximal utilization of intramuscularly (hereinafter "IM") -administered MLV IBR vaccines has been especially hampered by the hazards of vaccine-induced abortions. That is, abortion rates as high as 60% have been reported after IM administration of some MLV IBR vaccines (Kahrs, R. F., J. Am. Vet. Med. Assoc., 171:1055-1064 (1977); and Kendrick, J. W. et al, Am. J. Vet. Res., 28:1269-1282 (1967)). In addition, with the MLV IBR vaccines currently in use, there is the danger of reversion to virulence.
In a search for safer MLV IBR vaccines, specialized vaccines have been developed (Todd, J.D. et al, J. Am. Vet. Med. Assoc., 159:1370-1374 (1971); Kahrs, R. F. et al, J. Am. Vet. Med. Assoc., 163:427-441 (1973); Smith, M. W. et al, Can. Vet. J., 19:63-71 (1979); Zygraich, N. et al, Res. Vet. Sci., 16:328-335 (1974); and Kucera, C. J. et al, Am. J. Vet. Rest, 39:607-610 (1978)). These vaccines have been found to be immunogenic and safe for IN administration to pregnant cattle and can prevent abortions in pregnant cows which have been challenge-exposed to virulent IBRV. However, they have a disadvantage in that they can only be administered by the IN route. This is because, when administered IN, one such IBR vaccine replicates to a limited extent at the lower temperature of the upper respiratory tract. However, when administered IM, the vaccine replicates poorly or not at all at normal body temperatures (Zygraich, N. et al, Res. Vet. Sci., 16:328-335 (1974)). On the other hand, another IBR vaccine is insufficiently attenuated for IM administration to pregnant animals although safe when given IN (Todd, J. D., J. Am. Vet. Med. Assoc., 163:427-441 (1973)). Furthermore, some of the vaccine strains produce mild or moderate respiratory disease even after IN administration, and they do not prevent signs of IBR following field challenge exposure (Kahrs, R. F. et al, J. Am. Vet. Med. Assoc., 163:437-441 (1973)). Vaccination by temperature-sensitive viruses does not prevent the installation of virulent virus in a latent form or the reexcretion of the virulent virus. Moreover, vaccination with temperature-sensitive viruses does not inhibit reexcretion of a wild-type strain latently carried by animals at the time of vaccination (Nettleton, P. F. et al, In: Latent Herpesvirus Infections in Veterinary Medicine, Eds. G. Whittmann, R. M. Gaskell and H.-J. Rziha (Martinus Nijhoff Publishers: Boston/The Hague/Dordrecht), pages 191-209 (1984); Straub, O. C., Comp. Immun. Microbiol. Infect. Dis., 2:285-294 (1979); Thiry, E. et al, Veterin. Microbiol. 10:371-380 (1985); and Zuffa, A. et al, Zentrabl. Veterinaermed. Reihe B., 29:413-425 (1982)).
Accordingly, neither the IM-administered MLV IBR vaccines, which are unsafe for pregnant cows, nor the MLV IBR vaccines that must be administered IN, discussed above fit comfortably into many of the current management practices. That is, vaccination of large numbers of animals by the IN route is inconvenient and potentially dangerous to animal handlers. In addition, screening to identify pregnant animals prior to immunization is often not desirable or cost effective.
III. Attenuated Properties of Thymidine Kinase-Negative Herpesvirus Mutants
Recently, temperature-resistant, thymidine kinase-negative (hereinafter "tk.sup.- ") IBR vaccines derived from the thymidine kinase positive (hereinafter "tk.sup.+ "), i.e., wild-type, Los Angeles strain of IBRV (ATCC No. VR 188) have been developed which overcome many of the problems that have limited the use of currently available vaccines (Kit, S. et al, Virol., 130:381-389 (1983); Kit, S. et al, Arch. Virol., 86:63-83 (1985); Kit, S. et al, Vaccine, 4:55-61 (1986); and U.S. Pat. Nos. 4,569,840 and 4,703,011, which articles and patents are incorporated by reference herein in their entirety)). These IBRV vaccines consist of plaque-purified IBRV isolates that replicate equally well at either 39.1.degree. C. or 34.5.degree. C. in rabbit skin, bovine tracheal cells and bovine kidney cells. Hence, they are designed "temperature-resistant". This is in contrast to those IBRV strains that are designated "temperature-sensitive", that is, those used for the IN-administered vaccines which replicate only about 10.sup.-4 to 10.sup.-7 as well at 39.1.degree. C. as at 34.5.degree. C. In addition to the ability to replicate equally well at 39.1.degree. C. or 34.5.degree. C., the tk.sup.- IBR vaccines lack the ability to produce a functional thymidine kinase enzyme (hereinafter "TK") in infected cells. In one vaccine, designated IBRV(B8-D53) (ATCC No. VR 2066) (U.S. Pat. No. 4,569,840), the failure to produce a functional TK results from a mutagen-induced mutation. With a second vaccine, designated IBRV(NG)dltk (ATCC No. VR 2112) (U.S. Pat. No. 4,703,011), the failure to produce a functional TK results from a deletion of about 400 base pairs (hereinafter "bp") from the coding sequences of the IBRV tk gene. In addition to this deletion, there is an insertion into the IBRV tk gene of a 40 bp oligonucleotide, designated NG, with stop codons in all three reading frames. The characteristics, i.e., temperature resistance and tk.sup.-, directly contribute to the superiority of these IBRVs as vaccines.
As an alternative to the above-described tk.sup.- mutagen-induced mutants and tk.sup.- deletion mutants of IBRV, embodiments of the present invention were developed. Specifically, these embodiments relate to IBRV mutants that fail to produce any functional thymidine kinase as a result of an insertion in the coding region of the IBRV tk gene without deleting any nucleotide sequences from the IBRV tk gene. Insertion mutations inactivate the function of the IBRV tk gene by disrupting normal transcription and translation of the IBRV tk gene. Based on the findings that the previously described IBRV tk.sup.- mutagen-induced mutants and tk.sup.- deletion mutants are safe and efficacious vaccines against IBR disease, it is believed that the IBRV tk.sup.- insertion mutants of the present invention will also be useful as vaccines against IBR disease. The development of this embodiment of the present invention is based in part on the description of the location of the IBRV tk gene and the nucleotide sequence thereof, and the enrichment and selection procedures developed for isolating the IBRV tk.sup.- mutants described in U.S. Pat. Nos. 4,569,840 and 4,703,011. That is, prior to U.S. Pat. No. 4,703,011, it had not been possible to develop IBRV mutants that fail to produce any functional thymidine kinase as a result of an insertion in the IBRV tk gene because in the art (1) the location of the IBRV tk gene on the physical map of IBRV DNA was not known; (2) the approximate boundaries of the nucleotide sequences delineating the coding region of the IBRV tk gene were not known; and (3) the restriction nuclease sites within the IBRV tk gene to allow appropriate insertions to be made in a cloned IBRV tk gene were not known. In addition, prior to U.S. Pat. Nos. 4,569,840 and 4,703,011, it was not known in the art what selection drugs were advantageous for enrichment and selection procedures. As a result, prior to U.S. Pat. Nos. 4,569,840 and 4,703,011, there was insufficient information to engineer and isolate an insertion mutant in the IBRV tk gene.
IV. Distinguishing Vaccinated From Infected Animals
The temperature-resistance and tk.sup.- characteristics of the IBRV mutants discussed above greatly improves their safety and usefulness as vaccines. However, the dormancy feature of IBRV still makes it difficult to effect eradication of IBR through the application of quarantine measures which are intended to prevent the spread of IBR by the isolation of IBRV-infected herds and the slaughter of IBRV-infected animals. That is, with existing MLV IBR vaccines, it is impossible to determine, by simple blood tests, whether a specific animal, which does not show symptoms of illness, is a carrier of a dormant IBRV. This is because usage of most current vaccines masks infections. Hence, since animals which appear healthy may actually be carriers and, thus, spreaders of IBRV, it is important to be able, even after vaccination, to identify infected animals and herds so as to be able to apply quarantine measures.
In addition, some countries require that imported livestock, whether for breeding, for stocking of farms, or for market, be tested and shown not to be carriers of IBRV, i.e., the animals cannot be imported unless they are seronegative for IBRV. With current killed and MLV IBR vaccines, a producer who elects to protect his animals from the diseases which accompany the stresses of shipping, or who is forced by the circumstances of IBR infection in an endemic region to vaccinate susceptible animals, finds himself at a severe economic disadvantage. This is because vaccination of the stock with current killed and MLV IBR vaccines results in a positive serological test for IBRV. Revaccination to enhance protection further increases IBRV antibody titers. As a result, the farmer's ability to export valuable livestock and to sell his stock at home is restricted and he is at a disadvantage whether he vaccinates or does not vaccinate. Hence, an IBR vaccine is needed that can be administered safely, can protect animals from disease and dormant infections caused by field strains of IBRV, has a low or nonexistent probability of reversion to virulence, and yet, does not produce a positive serological test for IBRV. Such a vaccine would allow exportation of livestock and vaccination programs to be pursued unhindered by the fear of quarantine. A producer could then minimize losses within his own herd, while animal health authorities could continue with their respective control measures.
To meet the needs discussed above, e.g., the IBRV(NG)dltk vaccine described in U.S. Pat. No. 4,703,011 was further modified to produce vaccines, such as IBRV(NG)dltkdlgIII (ATCC No. VR 2181), described in U.S. patent application Ser. No. 116,197, filed Nov. 3, 1987, which U.S. application is incorporated by reference herein in its entirety. This IBR vaccine not only fails to produce any functional TK as a result of a deletion in the IBRV tk gene but also fails to produce any antigenic IBRV gIII polypeptides as a result of a deletion in the IBRV gIII gene. The IBRV gIII gene has been found to be a useful serological marker for IBRV infections.
V. Viral Based Vectors
During the last few years, recombinant DNA technology has permitted the construction of chimeric viruses which express genetic information from more than one origin.
A variety of unique viral based vectors have been developed such as adenovirus based vectors (Ruether, J. E. et al, Mol. Cell. Biol. , 6:123-133 (1986); and Haj-Ahmad, Y. et al, J. Virol., 57:267-274 (1986)) as well as herpes simplex virus based vectors (Smith, M. et al, Proc. Natl. Acad. Sci. USA, 81:5867-5870 (1984); and Smiley, J. R. et al, J. Virol., 61:2368-2377 (1987)), Herpes saimiri virus based vectors (Desrosiers, R. C. et al, Mol. Cell. Biol., 5:2796-2803 (1985)), baculovirus based vectors (van Wyke Coelingh, K. L. et al, Virol., 160:465-472 (1987); Miyamoto, C. et al, Mol. Cell. Biol., 5:2860-2865 (1985); Smith, G. E. et al, Proc. Natl. Acad. Sci. USA, 82:8404-8408 (1985); Smith, G. E. et al, Mol. Cell. Biol., 3:2156-2165 (1983); Matsuura, Y. et al, J. Gen. Virol., 68:1233-1250 (1987); and Kang, C. Y. et al, J. Gen. Virol., 68:2607-2613 (1987)), and nuclear polyhedrosis virus based vectors (Hu, S. -L. et al, J. Virol., 61:3617-3620 (1987); and Marumoto, Y. et al, J. Gen. Virol., 68:2599-2606 (1987)).
The use of viral based vectors as vaccines is advantageous over subunit vaccines because replication of the viral based vectors within the host amplifies the amount of foreign antigen being expressed thereby, often increasing both cell-mediated and humoral immunogenic responses to the foreign antigen. In addition, the expression of foreign antigens within infected cells of the immunized host provides for post-translational modifications of the foreign antigen and thus antigen presentation more closely resembling those occurring during natural infection with the pathogen from which the foreign antigen is derived. Further, the use of safe, live-attenuated virus as vectors allows for the immunization against the pathogen from which the foreign antigen is derived.
The most extensively developed and exploited viral based vectors are the vaccinia virus based vectors (Mackett, M. et al, Proc. Natl. Acad. Sci. USA, 79:7415-7419 (1982); Panicali, D. et al, Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); and Fuerst, T. R. et al, Mol. Cell. Biol., 7:2538-2544 (1987)). Vaccinia virus based vectors which have been described include those which are useful as vaccines for hepatitis B (Smith, G. L. et al, Nature, 302:490-492 (1983); and Cheng, K.-C. et al, J. Virol., 61:1286-1290 (1987)); rabies (Kieny, M. P. et al, Nature, 312:163-166 (1984); and Wiktor, T. F. et al, Proc. Natl. Acad. Sci. USA, 81:7194-7198 (1984)); malaria (Smith, G. L. et al, Science, 224:397-399 (1984)); human respiratory syncytial virus (King, A. M. Q. et al, J. Virol. 61:2885-2890 (1987); human parainfluenza virus type 3 (Spriggs, M. K. et al, J. Virol., 61:3416-3423 (1987)); rotavirus SA11 (Andrew, M. E. et al, J. Virol., 61:1054-1060 (1987)); influenza (Smith, G. L. et al, Proc. Natl. Acad. Sci. USA, 80:7155-7159 (1983); Smith, G. L. et al, Virol., 160:336-345 (1987)); herpes simplex virus (Paoletti, E. et al, Proc. Natl. Acad. Sci. USA, 81:193-197 (1984); Gillespie, J. M. et al, J. Clin. Microbiol., 23:283-288 (1986); and Sullivan, V. et al, J. Gen. Virol., 68:2587-2598 (1987)); vescicular stomatitis virus (Mackett, M. et al, Science, 227:433-435 (1985)); human immunodeficiency virus envelope proteins (Chakrabarti, S. et al, Nature, 320:535-537 (1986); Hu, S. -L. et al, Nature, 320:537-540 (1986); and Hu, S. -L. et al, Nature, 328:721-723 (1987)); and Friend murine leukemia virus envelope proteins (Earl, P. L. et al, Science, 234:728-731 (1986)). A vaccinia based viral vector which expresses the coding gene sequences of the PRV g92 gene has also been described (Robbins, A. K. et al, European Pat. Publication No. 0,162,738). In addition, vaccinia-based viral vectors which express host genes, such as murine interleukin-2 (Ramshaw, I. A. et al, Nature, 329: 545-546 (1987)) and murine Class I major histocompatibility complex antigen H-2K.sup.d, have been reported (Coupar, B. E. H. et al, Proc. Natl. Acad. Sci. USA, 83:7879-7882 (1986)).
Viral based vectors offer several significant advantages. First, immunization against more than one disease is possible with a single virus strain. This creates economy of production. Second, for many vaccines, two or more different modified-live viruses cannot be employed in combination in a single formulation because the replication of the viruses in the host causes mutual interference, and thereby impairs immunization. This situation is circumvented with viral based vectors because there is only a single replicating genome. Third, vaccines against causative agents of infectious disease, such as viruses and bacteria, for which vaccines have not previously been available or feasible, may for the first time be developed using viral based vectors which express the antigens of the causative agents of infectious disease. This is because the only component of the causative agents of infectious disease which is carried by the viral based vector is the gene coding for the antigen of the causative agent of disease which is responsible for eliciting immunity. Other components of the causative agents of infectious disease that are responsible for the pathobiology of the disease are eliminated from the viral based vector.
Although there is evidence that genetically engineered vaccinia viruses have reduced pathogenicity, there are several major obstacles to their general use as vaccines. First, severe complications can occur after vaccination, especially in immunodeficient individuals. Second, they are highly infectious for many animal species and humans. The insertion of genes from heterologous virus species into vaccinia-based viral vectors could alter the recombinant vaccinia virus host range or tissue tropism. Therefore, as agents, vaccinia virus based vectors pose potential health hazards. Third, there is no purpose served in vaccinating animals against smallpox, i.e., the disease that vaccinia protects against. Finally, potential recombination events between vaccinia and indigenous animal pox viruses might regenerate virulence of the vaccinia virus which will cause smallpox disease in humans.
IBRV-based viral vectors have distinct advantages over vaccinia-based viral vectors for the vaccination of cattle because they (1) protect cattle against the important bovine diseases of IBR and IPV; and (2) are host-limiting, i.e., they do not infect humans.
In embodiments of the present invention, IBRV has been utilized as the basis for viral based vectors. IBRV is a typical alpha herpesvirus with a genome consisting of linear double-stranded DNA molecules approximately 135-140 kilobases (hereinafter "kb") in size. Like vaccinia and like other herpesviruses, IBRV can tolerate nucleotide sequence deletions of 4 kb or more, and can tolerate foreign DNA insertions of 5-10 kb or more.
Prior to U.S. Pat. No. 4,703,011 and U.S. patent application Ser. No. 116,197, filed Nov. 3, 1987, now U.S. Pat. No. 4,992,051 it was not possible to develop IBRV-based viral vectors because there was no knowledge of the location of suitable, insertion sites, i.e., non-essential IBRV genes, which could be employed for the insertion of foreign genes. U.S. Pat. No. 4,703,011 and U.S. patent application Ser. No. 116,197, filed Nov. 3, 1987, however, identified the location of the IBRV tk and IBRV gIII genes on the physical map of IBRV DNA and demonstrated that they were not essential for virus replication in cultured cells. Hence, suitable insertion sites for foreign genes became known for the first time. Furthermore, U.S. Pat. No. 4,703,011 and U.S. patent application Ser. No. 116,197, filed Nov. 3, 1987, now U.S. Pat. No. 4,992,051 described for the first time the nucleotide sequences of the coding regions, the promoter regions, the translation start and stop signals, and the polyadenylation signals of the IBRV tk and IBRV gIII genes.
In the present invention, the isolation of IBRV DNA fragments containing IBRV gene promoters and the ligation of IBRV promoters to the coding nucleotide sequences of foreign genes has been described for the first time. Further, it has been determined for the first time in the present invention that in IBRV-based viral vectors, the foreign gene can be expressed by a foreign gene promoter and does not require an IBRV promoter for expression. Thus, for the first time in the present invention, it has been possible to develop IBRV-based viral vectors.